Nitric Oxide Production Activity Research Articles

Opioid receptor agonist Eribis peptide 94 reduces infarct size in different porcine models for myocardial ischaemia and reperfusion

Eribis peptide 94 (EP 94) is a novel enkephalin analog, thought to interact with the μ- and δ-opioid receptors. The purpose of the present study was to examine the cardioprotective potential of EP 94 in two clinically relevant porcine models of myocardial ischaemia and reperfusion, and to investigate if such an effect is associated with an increased expression of endothelial nitric oxide synthase (eNOS). Forty-one anesthetized pigs underwent 40min of coronary occlusion followed by 4h of reperfusion. In Protocol I, balloon occlusion of the left anterior descending artery was performed with concurrent intravenous administration of (A) vehicle (n=7), (B) EP 94 (1ug/kg) after 5, 12, 19 and 26min of ischaemia (n=4) or (C) EP 94 (1ug/kg) after 26, 33, 40min of ischaemia (n=6). In Protocol II, open-chest pigs were administered (D) vehicle (n=6) or (E) 0.2ug/kg/min of EP 94 (n=6) through an intracoronary infusion into the jeopardized myocardium, started after 30min of ischaemia and maintained for 15min. The hearts were stained and the protein content of eNOS measured. EP 94 reduces infarct size when administered both early and late during ischaemia compared with vehicle (infarct size group A 61.6±2%, group B 50.2±3% and group C 49.2±2%, respectively, P<0.05), as well as when infused intracoronary (infarct size group D 82.2±3.9% and group E 61.2±2.5% respectively, P<0.01). Phosphorylated eNOS Ser1177 in relation to total eNOS was significantly increased in the group administered EP 94, indicating activation of nitric oxide production.

Influence of cultivation conditions, season of collection and extraction method on the content of antileishmanial flavonoids from Kalanchoe pinnata

Leaves from Kalanchoe pinnata (Lamarck) Persoon (Crassulaceae) are popularly used for healing wounds. Its antileishmanial properties are established in experimental animals, and its active flavonoid components have been identified. In this study, we attempted to standardize the extract from K. pinnata leaves by evaluating the influence of season of harvest, sunlight exposure and method of extraction on antileishmanial flavonoids content. HPLC-DAD-MS was used to identify and quantify the active antileishmanial flavonoids in different extracts. ANOVA test for analyses of variance followed by the Tukey test of multiple comparisons were used in the statistical analysis. The antileishmanial potential was assessed by the activation of nitric oxide production by murine macrophage using the Griess method. We demonstrated that active flavonoids were significantly more abundant when the leaves were collected in the summer, and that aqueous extraction at 50°C allowed the highest flavonoid extraction. The benefit of sunlight exposure was confirmed in plants cultivated under direct sunlight when compared with those that grown under shade. Under sunny conditions the yield of the most active antileishmanial favonoid quercitrin was increased by 7-fold. All aqueous extracts tested were capable to enhance the macrophage nitric oxide production. However, hot aqueous extract from leaves collected in summer exhibited the higher activity, in agreement with HPLC-DAD-MS analysis tendency. In addition, with the aim of reducing the individual chemical variations of the plant constituents and optimizing the production of the active extract, it was obtained in vitro monoclonal KP specimens that were easily adapted to field conditions and were able to produce antileishmanial flavonoids. Our study reports the better conditions of cultivation, harvest and extraction protocol for obtaining a K. pinnata extract exhibiting the highest antileishmanial activity. Additionally, we propose the flavonoids quercetin 3-O-α-L-arabinopyranosyl (1→2)-α-L-rhamnopyranoside and quercitrin, as satisfactory chemical markers for standardization purposes.

Aloe-emodin is an interferon-inducing agent with antiviral activity against Japanese encephalitis virus and enterovirus 71

In this study, aloe-emodin was identified as a potential interferon (IFN)-inducer by screening compounds from Chinese herbal medicine. Aloe-emodin showed low cytotoxicity to human HL-CZ promonocyte cells and TE-671 medulloblastoma cells and significantly activated interferon-stimulated response element (ISRE) and gamma-activated sequence (GAS)-driven cis-reporting systems. Moreover, aloe-emodin upregulated expression of IFN-stimulated genes such as dsRNA-activated protein kinase and 2′,5′-oligoisoadenylate synthase. Aloe-emodin resulted in significant activation of nitric oxide production. The antiviral activity of aloe-emodin against Japanese encephalitis virus (JEV) and enterovirus 71 (EV71) was evaluated using dose- and time-dependent plaque reduction assays in HL-CZ cells and TE-671 cells. The 50% inhibitory concentration (IC 50) of aloe-emodin ranged from 0.50 μg/mL to 1.51 μg/mL for JEV and from 0.14 μg/mL to 0.52 μg/mL for EV71. Aloe-emodin showed clearly potent virus inhibitory abilities and achieved high therapeutic indices, in particular for HL-CZ cells. Therefore, the study demonstrated dose- and time-dependent actions of aloe-emodin on the inhibition of JEV and EV71 replication via IFN signalling responses.

Ergosta-4,6,8(14),22-tetraen-3-one from Vietnamese Xylaria sp. possessing inhibitory activity of nitric oxide production

From the methanolic extract of Xylaria sp. collected in Vietnam, a fluorescent constituent, ergosta-4,6,8(14),22-tetraen-3-one (1) was isolated and elucidated by the combination of 2D NMR, high resolution MS, IR and UV spectroscopy. Its inhibitory activity of nitric oxide production in RAW 264.7 cells stimulated by lipopolysaccharide was examined and showed a potential activity with the IC50 value of 28.96 µM.

Antileishmanial and antitrypanosomal activity of bufadienolides isolated from the toad Rhinella jimi parotoid macrogland secretion

Amphibian skin secretions are considered a rich source of biologically active compounds and are known to be rich in peptides, bufadienolides and alkaloids. Bufadienolides are cardioactive steroids from animals and plants that have also been reported to possess antimicrobial activities. Leishmaniasis and American Trypanosomiasis are parasitic diseases found in tropical and subtropical regions. The efforts toward the discovery of new treatments for these diseases have been largely neglected, despite the fact that the only available treatments are highly toxic drugs. In this work, we have isolated, through bioguided assays, the major antileishmanial compounds of the toad Rhinella jimi parotoid macrogland secretion. Mass spectrometry and 1H and 13C NMR spectroscopic analyses were able to demonstrate that the active molecules are telocinobufagin and hellebrigenin. Both steroids demonstrated activity against Leishmania (L.) chagasi promastigotes, but only hellebrigenin was active against Trypanosoma cruzi trypomastigotes. These steroids were active against the intracellular amastigotes of Leishmania, with no activation of nitric oxide production by macrophages. Neither cytotoxicity against mouse macrophages nor hemolytic activities were observed. The ultrastructural studies with promastigotes revealed the induction of mitochondrial damage and plasma membrane disturbances by telocinobufagin, resulting in cellular death. This novel biological effect of R. jimi steroids could be used as a template for the design of new therapeutics against Leishmaniasis and American Trypanosomiasis.

Cohaerins C—F, Four Azaphilones from the Xylariaceous Fungus Annulohypoxylon cohaerens.

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Inhibitory activity of nitric oxide production in RAW 264.7 cells of daldinals A–C from the fungus Daldinia childiae and other metabolites isolated from inedible mushrooms

Three benzophenone derivatives, daldinals A–C, from the Japanese fungus Daldinia childiae were evaluated for their inhibitory activity of nitric oxide (NO) production stimulated by lipopolysaccharide (LPS) in RAW 264.7 cells. They strongly suppressed the LPS-induced production of NO with IC50 values of 15.2, 4.6 and 6.4 μM, respectively. To clarify the mechanism involved, total RNA extraction, followed by reverse-transcribed, polymerase chain reaction (PCR) for inducible nitric oxide synthase (iNOS) mRNA and finally electrophoresis on agarose gel were performed. These experimental results suggest that the inhibition of the LPS-induced NO production of daldinal B is due to the inhibition of iNOS mRNA synthesis. Further, their biological activities were compared with those of other metabolites obtained during our studies on biologically active substances from inedible fungi in recent years.

Cohaerins C–F, four azaphilones from the xylariaceous fungus Annulohypoxylon cohaerens

Abstract Four azaphilones named cohaerins C–F, along with 4,5,4′,5′-tetrahydroxy-1,1′-binaphthyl were isolated from the methanolic extract of the stromata of Annulohypoxylon cohaerens (Ascomycetes, Xylariaceae). Cohaerins C–E constitute typical azaphilones, bearing a γ-lactone ring, while cohaerin F has an unprecedented carbon skeleton, lacking the lactone ring of the azaphilones and with an aliphatic side chain being attached directly to the azaphilone backbone by a C–C bond. Their structures were determined by 2D NMR, IR, UV, and CD spectroscopy. They showed moderate inhibitory activity of nitric oxide production in RAW cells, and strong and nonselective antimicrobial effects.

Flavonol glycosides from Costus spicatus

Two flavonol diglycosides, tamarixetin 3-O-neohesperidoside, kaempferide 3-O-neohesperidoside and the known quercetin 3-O-neohesperidoside, together with six other known flavonoids were isolated from the leaves of Costus spicatus and their structures were elucidated by a combination of spectroscopic and chemical methods. The flavonol diglycosides were evaluated for inhibitory activity of nitric oxide production by activated macrophages (Fig. 1 ).

Endothelial nitric oxide synthase expression in tumor vasculature is correlated with malignancy in human supratentorial astrocytic tumors.

Endothelial nitric oxide synthase (eNOS) may play an important role in the regulation of tumor blood flow and vascular permeability. However, there have been no reports describing alterations of eNOS expression in relation to malignant progression in human astrocytic tumors. We immunohistochemically studied the relationship between eNOS expression in tumor vasculature and malignancy in supratentorial astrocytic tumors. Tissue samples were obtained from 12 patients with low-grade astrocytomas, 10 with anaplastic astrocytomas, and 17 with glioblastomas. Normal brain tissue samples were obtained from four patients with other brain diseases. Immunohistochemical staining was performed using the avidin-biotin complex method, with polyclonal anti-eNOS antibody, and the levels of eNOS expression in endothelial cells were evaluated as slight, moderate, or intense on the basis of eNOS immunoreactivity. The proliferative potential was assessed as the MIB-1 staining index for tumor cells. The expression of eNOS was slight in all specimens of normal brain tissue, slight in 7 and moderate in 5 specimens of low-grade astrocytoma, slight in 2, moderate in 6, and intense in 2 specimens of anaplastic astrocytoma, and moderate in 5 and intense in 12 specimens of glioblastoma. The MIB-1 staining index (mean+/-standard deviation) was 0.2+/-0.2% for normal specimens, 1.8+/-0.6% for low-grade astrocytomas, 9.6+/-6.9% for anaplastic astrocytomas, and 18.5+/-7.7% for glioblastomas. The MIB-1 staining indices for slight, moderate, and intense eNOS expression were 2.0+/-2.3%, 10.8+/-9.8%, and 16.9+/-7.7%, respectively. Expression of eNOS in tumor vessels was significantly correlated with histological grade and proliferative potential. These findings suggest that astrocytic tumor vessels possess higher activity for nitric oxide production than do normal vessels.

Lymphotoxin inhibits Chlamydia pneumoniae growth in HEp-2 cells.

Cytokines such as gamma interferon and tumor necrosis factor alpha (TNF-alpha) inhibit the intracellular replication of Chlamydia pneumoniae or Chlamydia trachomatis. In this study, we found that another cytokine, lymphotoxin (TNF-beta), restricts the growth of C. pneumoniae in HEp-2 cells. When lymphotoxin (10 U/ml) was added during incubation from 8 to 16 h postinoculation, inclusion body formation was severely reduced. In addition, we observed activation of nitric oxide production and the nuclear transition of NF-kappaB in HEp-2 cells in response to lymphotoxin. These results suggest that inhibition of chlamydial growth by lymphotoxin is mediated, at least in part, by nuclear transition of NF-kappaB, resulting in induction of nitric oxide synthase to produce nitric oxide, a potent bacteristatic agent. This is the first report on antichlamydial activity of lymphotoxin through induction of nitric oxide.

Intracellular localization and activation of endothelial nitric oxide synthase.

Nitric oxide production by the Ca2+/calmodulin-dependent enzyme endothelial nitric oxide synthase primarily reflects changes in intracellular [Ca2+], increasing as Ca2+ rises and decreasing as Ca2+ falls. This simplistic bimodal mechanism of regulation has recently been refined by the finding that the binding of Ca2+/calmodulin to endothelial nitric oxide synthase involves the disruption of the association of endothelial nitric oxide synthase from the scaffolding protein caveolin and the subsequent translocation of the enzyme from plasmalemmal caveolae. Moreover, other endothelial nitric oxide synthase-associated proteins could account for a delayed Ca(2+)-independent activation of nitric oxide production, and may be involved with caveolin in the reversible trafficking of a large endothelial nitric oxide synthase-containing heterocomplex between the caveolae and cytosolic cell structures.